We examined acute alterations in gait and oxygen cost from shod-to-barefoot running in habitually-shod well-trained runners with no prior experience of running barefoot. Thirteen runners completed six-minute treadmill runs shod and barefoot on separate days at a mean speed of 12.5 km·h-1. Steady-state oxygen cost in the final minute was recorded. Kinematic data were captured from 30-consecutive strides. Mean differences between conditions were estimated with 90% confidence intervals. When barefoot, stride length and ground-contact time decreased while stride rate increased. Leg-and vertical stiffness and ankle-mid-stance dorsi-flexion angle increased when barefoot while horizontal distance between point of contact and the hip decreased. Mean oxygen cost decreased in barefoot compared to shod running (90% CI -11% to -3%) and was related to change in ankle angle and point-of-contact distance, though individual variability was high (-19% to +8%). The results suggest that removal of shoes produces an alteration in running gait and a potentially-practically-beneficial reduction in mean oxygen cost of running in trained-habitually-shod runners new to running barefoot. However, high variability suggests an element of skill in adapting to the novel task and that caution be exercised in assuming the mean response applies to all runners.